OUR SPECIFIC OBJECTIVES FOR THIS PAST YEAR: 1) With respect to PLD, previous studies revealed that PLD2 co-localized with components of specialized lipid microdomains in the plasma membrane sometimes "lipid rafts". We have now investigated whether PLD itself is required for the functional integrity of "lipid rafts" because localized production of phosphatidic acid and diacylglycerol through PLD could have a substantial impact on membrane dynamics. 2) We have also investigated the role of PLD in the activation of sphingosine kinase (SK) because PLD products are potential activators of SK and SK has been implicated in the generation of a calcium signal. 3) As noted in the Introduction, we believed that Ca2+-specific CRAC was not the only mechanism for the entry of Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells. The transient receptor potential cannonical (TRPC) channels were originally considered as candidates for CRAC but their electrophysiological properties did not fit with those of CRAC and some TRPCs were known to conduct Sr2+ and other divalent metal ions. As described last year, we obtained strong evidence that TRPCs interact with the recently identified CRAC components, Orai1 and its Ca2+ sensor STIM1. We have examined this interaction at a bichemical and cellular level. RESULTS: 1) PLD AND LIPID RAFT FUNCTION: Suppression of PLD function, either with primary butanol, which subverts production of phosphatidic acid to the inert phosphatidylbutanol, or siRNAagainst PLD2 and to a much lesser extent PLD1 results in dispersal of lipid raft components including LAT, Thy1, and GPI. Once mast cells are activated, the translocation of the IgE receptor (FceRI) and its associated tyrosine kinases, Lyn and Syk, into lipid rafts as well as downstream phosphorylation events and degranulation are also blocked. Consistent results were obtained when cells were examined by use of fluorescent tagged molecules and confocal microscopy or by membrane fractionation techniques. These techniques indicated that tagged PLD2 also colocalizes with lipid raft constituents, a process that is prevented by the lipid raft dispersing agents. These effects were accompanied by a changes in distribution of the lipid raft component, ganglioside 1, in the plasma membrane as determined by binding of fluorescent-tagged cholera toxin B-subunit and confocal microscopy. These and earlier studies suggest that PLD2 activation is not only dependent on lipid raft integrity but also that PLD activity ensures functionality of lipid rafts (Bibliography, Ref. 2). 2) REGULATION OF CALCIUM MOBILIZATION AND DEGRANULATION BY PLD AND SPHINGOSINE KINASE (SK): In addition to being the primary source diacylglycerides for the activation of diglyceride-dependent isoforms of PKC(our studies, J. Immunol. 174:5201,2005), PLD1 is reported to regulate activation of SK1 and thus production of sphingosine 1-phosphate which, acting in conjunction with IP3, activates store-operated Ca2+ entry (SOCE)in stimulated mast cells. However, we find that knockdown of PLD2 or SK2, and not PLD1 or SK1, results in only a modest reduction of Ca2+ entry. Also contrary to previous reports, we find that this reduction is not unique to antigen stimulation as it is observed with pharmacologic stimulants such as thapsigargin and ionomycin. Doubts about the existence and physiologic significance of a PLD/SK/Ca2+ pathway were butressed in studies with bone-marrow derived mast cells (BMMC) from knockout mice. Deficiency in any one of the SK and PLD isoforms resulted in no substantial impairment in calcium signal. However, we did observe in two out of five experiments with SK2-deficient BMMC, defective migration of the Ca2+-sensor, STIM1, from the endoplasmic reticulum (the source of intracellular Ca2+) to the plasma membrane where STIM1 interacts with and activates Orai1 or, as we believe a heteromeric TRPC/Orai1 complex that constitutes the Ca2+ channel (more in item 3 below). Our conclusion at this stage of our studies is that SK deficiency results in disfunctional membrane trafficking (the best studied role of SK)and that the effects on calcium signaling, if any, are secondary. 3) IDENTIFICATION OF CALCIUM CHANNELS AND REGULATORY PROTEINS FOR CALCIUM ENTRY: As noted in the Introduction, depletion of intracellular stores of Ca2+ in the endoplasmic reticulum activates influx of external Ca2+ (SOCE) via the well characterized Ca2+-specifc current CRAC. CRAC has now been attributed to the interaction of a Ca2+-sensor, STIM1, in the endoplasmic reticulum and the Ca2+-specific channel protein Orai1. We found that overexpression of STIM1 and Orai1 allows entry of Ca2+ but not Sr2+ in stimulated mast cells. Knock down of individual TRPCs with siRNAs indicated that entry of Ca2+ and Sr2+ as well as degranulation was dependent on TRPC5 and to a lesser extent TRPC1. These and similar experiments suggested that TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca2+ or Sr+ to generate a signal for degranulation (J. Immunol. 180:2233, 2008). We have since observed similar scenarios in both tumor and primary mast cell lines of rodent and human origin and have confirmed the requirement for TRPC channels in BMMC from TRPC -/- mice. These mice exhibit attenuated allergic reactions (personal communication, Alasdair M. Gilfillan, LAD, NIAID) and BMMC derived therefrom exhibit defective a calcium signal, degranulation and much reduced production of inflammatory cytokines (studies in collaboration with Alasdair M. Gilfillan). Our findings on participation of TRPC channels bridge seemingly paradoxical observations of non-selective uptake of divalent metal ions by stimulated mast cells (early sudies by us and other workers) and highly selective uptake of Ca2+ through CRAC/Orai1 (recent studies by others) (Bibligraphy, Ref 3). Most recently, it has become apparent that initiation of the calcium signal is absolutely dependent of TRPC channels and that this occurs in discrete regions (cell protuberances) and patterns whereby regional flickers merge into propagating waves (Bibliography, Ref. 4). There is thus much more to be learnt about the fundamentals of calcium signal which is an essential signal for many types of cellular activities.